In professor Warren Grayson’s lab, we created 3D maps that allowed us to see the distribution of blood vessels and stem cells throughout the mouse skull for the first time. To do this, we developed a light-sheet microscopy platform that enabled us to image the entire top portion of the skull at single-cell resolution. Using our 3D maps, we discovered that stem cells were spatially associated with specific types of blood vessels during skull bone growth and healing. These results will help inform the research and development of regenerative therapies for patients suffering from large skull bone injuries.

Over the last 20–30 years, the field has gained a detailed understanding of the molecular composition of glutamatergic synapses, the site of excitatory neurotransmission in the central nervous system. To date, however, we know very little about cell type-specific differences in the molecular basis of glutamatergic synapse function. I’ve been working in Richard Huganir’s laboratory in the Department of Neuroscience, where my research has focused on uncovering molecular specializations at glutamatergic synapses received by inhibitory interneurons — minority cell types in cortical and hippocampal circuits. We believe this research is important as it could lay the foundations for new cell type-specific interventions to regulate the activity of subsets of glutamatergic synapses.

The temporal bone houses an incredible amount of tiny, geometrically complex anatomical structures that are important for hearing and balance. Surgical access in this region requires drilling through varying densities of bone and identifying surgical landmarks to avoid damaging hidden critical anatomy. Due to these inherent limitations in visibility and maneuverability, temporal bone surgery poses a risk for accidental damage to surrounding anatomy, which can cause hearing loss, vertigo, altered taste sensation and facial paralysis. One possible approach for mitigating accidental damage to surrounding structures is using intraoperative image-guided robotic systems that can determine the location of robotically controlled instruments relative to patient imaging and enforce safety barriers around contacting critical anatomy. A key obstacle to utilizing the full potential of these technologies is the lack of streamlined methods for labeling critical anatomy on patient CT imaging. While manually segmenting surgically relevant landmarks on preoperative imaging can be performed, it is extremely time intensive and prone to inter-reader variability. To overcome these limitations, I have developed an efficient, accurate and automated pipeline for segmenting structures in temporal bone CT scans under the guidance of Dr. Francis Creighton and Dr. Russell Taylor in the Laboratory for Computational Sensing + Robotics. This automated pipeline has the potential to provide robust anatomical information for developing immersive virtual surgical simulators, patient-specific anatomical models, population-based shape analyses of the temporal bone and improved guidance for surgical navigation systems.

Decades of research in cellular therapies for cancer have focused on modulation of cytotoxic — CD8+ — T cells, the immune system’s professional killers. In the Schneck lab, we have sought to advance these cell therapies to the clinic through development of acellular platforms that promote CD8+ T cell antitumor activity. That said, a recent appreciation for the pivotal role that helper — CD4+ — T cells play in therapeutic cancer immune responses has motivated us to generate nanoparticle technologies targeting both effector and helper functions of CD4+ T cells. In our recent work, we showed that our nanoparticles can expand rare antigen-specific murine and human CD4+ T cells. Intriguingly, unlike with other traditional stimulation platforms, CD4+ T cells activated with these nanoparticles demonstrate cytotoxic activity, a phenotype that has been observed almost exclusively in vivo, allowing us to probe the etiology of this uncommon but clinically relevant cell subset. Additionally, using a nanomaterial approach to spatially control the proximity of CD4+ and CD8+ T cells during activation, we demonstrated that help signals from CD4+ T cells could be relayed to CD8+ T cells, in turn enhancing CD8+ T cell memory formation, function, cytotoxicity and antitumor activity. These findings illustrate several ways in which CD4+ targeted nanotechnologies can bolster current approaches to cancer immunotherapy.

I did my research in the laboratory of Dr. Joel Blankson, where we study immune responses to viral infections. Part of my dissertation research involved characterizing T cell responses to endemic coronaviruses and SARS-CoV-2 following natural infections and vaccinations. In early 2020, it was known in the field that some COVID-19 unexposed individuals had T cell responses to SARS-CoV-2 despite never experiencing the virus. These preexisting immune responses might partially explain the divergent outcomes seen following COVID-19 infections. Work from our lab and others has shown that these preexisting T cell responses are partially a result of cross-reactive T cells that were primed by endemic human coronaviruses but can also recognize and respond to SARS-CoV-2. In my recent work, I show that COVID-19 mRNA [messenger RNA] vaccinations also enhance T cell responses to endemic coronaviruses. Additionally, I identified a conserved immunodominant epitope found in human and bat coronaviruses and demonstrate that vaccinated individuals are able to mount cross-reactive T cell responses to this epitope. Our work expands our understanding of cross-protective T cell epitopes and informs the development of vaccine strategies that, it is hoped, will elicit cross-protection across many coronaviruses.

In Hal Dietz’s laboratory, I focused my doctoral research on vascular Ehlers-Danlos syndrome [vEDS] — a rare genetic disorder due to collagen type III mutations that leads to spontaneous vascular rupture and dissection. It is widely believed that the often fatal vascular rupture occurs because of weak tissue in the vessel wall, due to reduced amounts of collagen. However, by uncovering genetic and environmental modifiers of disease, we discovered that signaling abnormalities in the endothelin signaling pathway [ET1/PLC/PKC/ERK] drive vascular rupture risk. Pharmacologic agents that inhibit this pathway prevent death from vascular rupture. These discoveries illustrate the power of complementary discovery-based methods to elucidate the molecular basis of disease, reveal novel understandings of vascular biology and discover new therapies for catastrophic vascular disease. Based on these results, the first clinical trial for patients with vEDS was initiated in 2021 using a novel PKCβ inhibitor, enzastaurin.

My research focuses on elucidating memory B cell and antibody responses following hepatitis C virus [HCV] or SAR-CoV-2 infection. The COVID-19 pandemic has reaffirmed the importance of vaccines in containing spread of disease and preventing serious illness. To do so, many vaccines elicit memory B cells specific for the receptor binding domains, which in turn produce durable and potent broadly neutralizing antibodies [bNAbs] against the virus. Unlike SARS-CoV-2, we currently do not have an approved vaccine for HCV. There are approximately 71 million people, many asymptomatic, living with chronic HCV infection that can lead to liver failure and hepatocellular carcinoma. One of the reasons we don’t have a vaccine is because we have not determined the critical epitopes that a vaccine can target. In the laboratory of Dr. Justin Bailey, we study how the adaptive immune system responds to HCV or SARS-CoV-2 infection with a focus on B cell/ antibody responses and their viral targets or escape mechanisms. For my thesis work, I developed a highly specific and sensitive method that captures HCV specific B cells. For the first portion of the project, I characterized memory B cells that are associated with low antibody levels during HCV infection. I found that plasma anti-HCV antibody levels were positively correlated with frequencies of resting and activated memory B cells. Additionally, anti-HCV antibody levels were negatively correlated with levels of the expression of FCRL5 on resting and PD-1 on activated memory B cells. For the second portion of the project, I isolated and characterized 55 cross-reactive HCV specific bNAbs from an elite HCV neutralizer who naturally cleared three infections. We discovered that HCV specific bNAbs use a wide range of antibody gene segments and they acquire similar mutations that are critical for binding to the virus, pointing to convergent co-evolution of multiple bNAb lineages. Epitope mapping and crystal structures revealed that HCV bNAbs target conserved and undescribed epitopes on the virus. These findings show which epitopes are critical for bNAbs to target on an HCV virion — important information in the development of an HCV vaccine.

I joined the cancer genomics lab led by Victor Velculescu and Rob Scharpf during my two years of research fellowship. During that time, my colleagues and I discovered and validated a new way to noninvasively diagnose patients with brain cancer and lung cancer via a blood test. This discovery is important as up until now, brain cancer could not be detected in the blood in the majority of the cases with any known methodologies. In lung cancer, we showed that detection at early stages is possible in a significant number of cases. Using our methodology, we were able to noninvasively subtype lung cancer into the two main categories: non-small cell versus small cell lung cancer.

One research interest of the Ewald lab is to understand how cancer cells are able to travel within the body to form distant metastasis. For my research, I focused on triple negative breast cancer [TNBC], an aggressive subtype of breast cancer typically associated with metastasis and worse patient outcomes. Patients with TNBC tumors express mesenchymal markers often associated with the epithelial to mesenchymal transition [EMT]. However, the role of EMT during spontaneous TNBC metastasis in vivo remains incompletely understood, and the clinical relevance of EMT in patients with TNBC is unclear. Using a combination of in vivo and ex vivo organoid assays, I demonstrated that EMT is required for TNBC metastasis formation. Importantly, my research highlighted the complexity of cancer cells’ states within TNBC metastases and revealed that the majority of TNBC metastases maintain some mesenchymal features. These results suggest that TNBC will be an important context in which to evaluate anti-EMT therapeutic strategies. Indeed, targeting cancer cells expressing mesenchymal markers would potentially inhibit both invasion and metastases development.

Schizophrenia [SZ] and bipolar disorder [BP] are highly heritable major psychiatric disorders that share a substantial portion of genetic risk as well as their clinical manifestations. This raises a fundamental question of whether, and how, common neurobiological pathways translate their shared polygenic risks into shared clinical manifestations. In the lab of Dr. Akira Sawa, we showed the miR-124-AMPAR pathway as a key common neurobiological mediator that connects polygenic risks with behavioral changes shared between these two psychotic disorders. We discovered upregulation of miR-124 in biopsied neuronal cells and postmortem prefrontal cortex from patients with SZ and BP. Intriguingly, the upregulation is associated with the polygenic risks shared between these two disorders. Seeking mechanistic dissection, we generated a mouse model that upregulates miR-124 in the medial prefrontal cortex, which includes brain regions homologous to subregions of the human prefrontal cortex. We demonstrated that upregulation of miR-124 increases GRIA2- lacking calcium permeable-AMPARs and perturbs AMPAR-mediated excitatory synaptic transmission, leading to deficits in the behavioral dimensions shared between SZ and BP.

The iris — the colorful tissue that rings the pupil — plays an important role in visual function by controlling pupil diameter to regulate the amount of light entering the eye. It is also a site of diverse ophthalmologic diseases and a potential source of cells for ocular auto-transplantation. In the laboratory of Dr. Jeremy Nathans, my research focuses on deciphering the cell types of the mouse iris and their genomic response to pupil dilation. More specifically, our work has (1) defined all of the major cell types in the mouse iris, (2) discovered two types of iris sphincter cells and two types of stromal cells, (3) revealed the differences in cell type-specific transcriptomes in the resting and dilated states, (4) clarified the neural crest contributions to the iris by using the Cre-loxP system. These findings expand on our fundamental understanding of the iris and should be a valuable reference for investigations of iris development, disease and pharmacology, for the isolation and propagation of defined iris cell types, and for iris cell engineering and transplantation.

Many cancers can be cured by surgery and systemic therapies when detected while they are still localized, yet most cancer types lack noninvasive screening modalities to identify them before they have metastasized to distant sites. Under the mentorship of Bert Vogelstein within the Ludwig Center at Johns Hopkins, my work focused on the development of a noninvasive blood-based diagnostic for the detection and localization of a variety of cancer types. This test, called CancerSEEK, assays the levels of circulating proteins and mutations in cell-free DNA. In a study applying this test to 1,005 patients with nonmetastatic, clinically detected cancers of the ovary, stomach, pancreas, esophagus, colorectum, lung or breast, CancerSEEK tests were positive in a median of 70% of the eight cancer types. Importantly, the specificity of CancerSEEK was greater than 99% when evaluated in a cohort of 812 healthy controls. Additionally, to further improve the performance of CancerSEEK, I developed a novel technological methodology that can more sensitively and specifically detect ultrarare circulating tumor DNA molecules. In summary, this work lays the conceptual and practical foundation for a single, multianalyte blood test for cancers of many types.

I work in the lab of Dr. Jie Xiao in the Department of Biophysics and Biophysical Chemistry. In general, we study how bacteria organize several proteins in the right time and place for successful cell division through super-resolution microscopy. My project in particular studies how a bacterial cytoskeletal protein, FtsZ, can act like a linear motor to drive the movement of cell wall synthases in the model bacterium Escherichia coli. This discovery is important because the proteins that synthesize bacterial cell walls are antibiotic targets, so understanding how cell wall synthases are distributed, activated and spatiotemporally regulated provides insight on potential future therapeutics.

The emergence of immunotherapy as an important tool in the fight against cancer takes advantage of the exquisite specificity of antibodies. Targets, however, are limited to those on the cell surface, while most driver mutations occur in the genes encoding intracellular proteins. To overcome this limitation, antibodies can be engineered to target peptides derived from mutant proteins that are presented on the cell surface by major histocompatibility complex class I [pMHC-I]. My studies conducted in the Gabelli lab elucidated the structural basis for antibody recognition to MHC-presented neoantigens to garner potent “off the shelf” therapeutics. Specifically, we have designed and developed two antibody bispecifics, called H2 and V2, that target a peptide derived from the tumor suppressor gene TP53 R175H mutation and the oncogene KRAS G12V mutation, respectively. Binding kinetics experiments revealed the two bispecifics bind their respective mutant pMHC with different kinetics, suggesting different modes of binding. The structure of the p53R175H-pMHC bound to the H2-Fab fragment showed that the H2 antibody formed a cage-like configuration around the p53R175H peptide, trapping the mutant histidine [His175] and the adjacent arginine [Arg174] residues in a stable interaction, providing the structural basis for the specificity. In contrast, the structure of the KRASG12V-pMHC bound to the V2-IgG showed a very hydrophobic interaction and a conformational change upon binding, highlighting the specificity. Notably, the two antibody fragments, in a bispecific format, induced a potent and specific T cell response. Our detailed structural understanding of the mechanisms of specificity allows for the development of more effective therapeutics. By exploiting the MHC-I presentation of neoantigens, we have achieved the first step toward a precision off-the-shelf medicine therapeutic that selectively targets mutated driver genes.

For the duration of the DDP [Johns Hopkins University Doctoral Diversity Program], I am conducting research full-time in Dr. Sandra Gabelli’s lab, where one of my projects involves studying NUDIX hydrolases, a superfamily of enzymes known for their ability to remove mutagenic nucleotides from the nucleotide pool. They are named after their shared ability to catalyze the hydrolysis of nucleoside diphosphate linked to a moiety X, hence the name NUDIX. Many NUDIX hydrolases have cellular roles ranging from the degradation of mRNA and processing of ADP-ribosylation to the removal of mutagenic nucleotides from the nucleotide pool. Though a highly conserved signature motif of 23 amino acids, G^N₁[5X]E^N₇[7X]R^N₁₅E^N₁₆XXE^N₁₉E^N₂₀XG^N₂₂U, known as the NUDIX signature sequence, allows us to identify these enzymes, more information is necessary to classify these enzymes into families. Previously, the NUDIX family represented by NudI was identified to be nucleoside triphosphatases with a preference for pyrimidine deoxynucleoside triphosphates. Recent studies have shown that NudI preferentially hydrolyzes geranyl pyrophosphate [GPP] instead of a nucleoside containing metabolite. I am characterizing an atypical NUDIX family, represented by NudI whose preferred substrate, geranyl pyrophosphate, lacks a nucleotide in comparison to the archetypical substrate. Using the conformational changes that NUDIX enzymes undergo upon substrate binding, product release and inhibitor binding will allow me to establish the rules of recognition. The characterization of NudI as a hydrolase of GPP, with its structural determinants of specificity and inhibition, is a step toward identifying the pathway it is involved in and establishing the rules for classifying NUDIX enzymes into families.

Breast microcalcifications, indicative of Ca2+ dysregulation, are early signs of breast cancer. My work highlights that the therapeutic targeting of calcium signaling is not straightforward. Since both elevation and depletion of Ca2+ levels can drive malignant phenotypes, the specific molecular mechanisms driving these changes need to be clearly understood. This is exemplified by my work on the opposite roles of Golgi/secretory pathway Ca2+-ATPase isoform SPCA2 in breast cancer subtypes. I showed that high expression of SPCA2 in receptor positive breast cancer confers poor survival prognosis and drives pro survival and chemoresistance. In triple negative breast cancer, I showed that low expression of SPCA2 confers poor survival prognosis, drives metastasis and drug resistance. Thus, the targeting of Ca2+ signaling in breast cancer needs to be subtype and isoform specific. Inhibitors that decrease SPCA2-driven Ca2+ signaling in luminal and HER2+ breast cancers and enhancers that reactivate deficient SPCA2-mediated calcium signaling in basal/TNBC could be effective therapeutic tools.

I study the significance of phospholipids in mitochondria in the Claypool lab. My current focus is on cardiolipin — a signature phospholipid that ensures cellular energy production via oxidative phosphorylation. Cardiolipins have been evolutionarily found in the structures of the ADP/ATP carrier, which exchanges ADP and ATP across the membrane to enable oxidative phosphorylation. Our recent work revealed that the cardiolipins within the ADP/ATP carrier support its structure and activity. This finding highlights the conserved important roles of specific lipid-protein interactions in mitochondrial biology.

My research focuses on the mechanisms and impact of citrate delivery to the skeleton. The Clemens lab focusses on understanding the processes of osteoblast bioenergetics and how this is involved in the formation of bone. By reading old research papers [from the 1940s], my mentor discovered that 80% of our total body citrate is stored in the skeleton. However, nobody knew how it got there and which role it played. We now discovered that osteogenic expression of the citrate transporter SLC13A5 plays a significant role in citrate partitioning into bone. A malfunctioning transporter significantly impacts bone mass and strength, and causes hypomineralization of teeth in both mice and humans. Understanding that osteoblast bioenergetics is not only important for the generation of energy but also for providing critical metabolites, such as citrate, to facilitate bone mineral formation is a new concept to the field. This finding is immediately relevant to developing better strategies for not only improving bone mass but also bone quality in metabolic diseases such as osteoporosis and diabetes or in rare diseases (e.g., SLC13A5 disease).

Building on the observation that a protein called NPTX2 [neuronal pentraxin 2], which is related to memory consolidation, is reduced in the CSF of schizophrenia patients, I revealed that its trafficking at the synapses is impaired in schizophrenia-relevant behaviors. A separate but related discovery I made is that in vivo NPTX2 synaptic trafficking is contingent on the connectivity status of pyramidal to PV-interneuron, which gates a form of visual critical period plasticity, which is seen during development. I am doing research in Dr. Paul Worley’s lab at the department of Neuroscience.

There is still no cure for the 38 million people worldwide living with HIV-1, due to the persistence of the virus in a latent reservoir of CD4+ T cells. Efforts to cure HIV-1 infection by reducing the size of this reservoir have focused on the shock and kill strategy, which relies upon inducing viral gene expression and revealing infected cells to the immune system (shock). Infected cells can then be targeted for destruction by immune cytolytic cells (kill). While some chemical agents have induced slight increases in HIV-1 gene expression, reductions in the size of the latent reservoir have not been observed. Critical to the success of shock and kill, then, is the development of novel kill strategies that can enhance the destruction of infected cells. My research in the labs of Drs. Robert and Janet Siliciano and Dr. Scheherazade Sadegh-Nasseri focused on how HIV-1 is recognized by the immune system and whether we could generate reagents that could promote recognition and killing of infected cells. This highly team-based study led to the discovery and production of bispecific antibody engagers that are exquisitely specific and sensitive to minute quantities of HIV-1 peptides presented on the surface of infected cells. These bispecific antibodies, with domains specific for both HIV-1 and cytolytic T cells, enabled robust killing of HIV-1 infected cells, in a manner dependent on the quantity of HIV-1 peptides presented on the infected cell. Given the many millions of individuals affected by this incurable disease, understanding mechanisms that could guide improved immunotherapies for cure strategies is essential.

Despite decades of molecular study, many of the earliest events in the development of human cancers remain unexplored. For example, nearly a third of human tumors undergo whole genome duplication [WGD] that can increase cancer cell fitness, but it is not known how or when these tetraploid cancers arise. This is because cancer onset, or oncogenesis, is unpredictable and may occur in just one or a few cells in an otherwise healthy tissue. In the Regot lab, we use live imaging of biosensors to dissect the signaling events that underlie these rare events. This approach allowed me to observe the effects of cancer mutations on cell signaling and the cell cycle. I found that oncogenic WGD results from inappropriate inactivation of cell cycle machinery and mitosis skipping. These skipped cells are primed for reentry into the cell cycle, despite having double the normal DNA content. I found that the same common mutations are associated with WGD in human lung adenocarcinomas and demonstrated that these oncogenes can cause WGD in primary human airway cells. These findings revealed the mechanism and timing of a key event in oncogenesis, whole genome duplication, which is a turning point in the development of aggressive cancers.

During development, the brain generates activity spontaneously, without any stimulus from the outside world. This activity trains the brain to be ready for sensory input and is important for the maturation of brain circuits. My research in the Bergles lab has uncovered the involvement of astrocytes, a glial cell type in the brain, which were often thought to be merely supporting cells. Using advanced imaging and molecular techniques, I have found that astrocytes and neurons coordinate spontaneous activity during early development of the auditory system, and that this activity likely mediates the maturation of both cell types. These results position astrocytes as potential therapeutic targets for disorders that involve the maturation of brain circuits, such as autism spectrum disorders and schizophrenia.

DNA fluorescence in situ hybridization [DNA FISH] allows for visualization of specific DNA sequences inside the cells, so it is a powerful tool for studying chromatin conformations and protein-genomic DNA interactions. Conventional DNA FISH requires global genome denaturation by high temperature, which is very harsh and may disrupt some heat-labile structures. To overcome this limitation, I developed a physiological-temperature DNA FISH method called genome oligopaint via local denaturation FISH [GOLD FISH]. GOLD FISH uses an enzymatic approach to locally denature genomic DNA, avoiding the harsh treatment in conventional DNA FISH. GOLD FISH can efficiently label both repetitive and nonrepetitive DNA sequences in cultured cells and tissue sections, allowing for studying chromatin conformational changes and cancer-relative copy number variations. In addition, GOLD FISH has single-nucleotide sensitivity, which can detect pathological point mutations and base-editing events in pathological samples. Taken together, the method I developed in the Dr. Taekjip Ha’s lab can facilitate both basic research and clinical diagnosis.

Uniquely among mammalian organs, skin is capable of dramatic size change in adults, making it ideal for studying size control mechanisms and regenerative medicine. The remarkable capacity of adult skin to grow under constant stretch is utilized clinically for reconstructive purposes in a process named tissue expansion, yet the mechanisms are unclear. In an established tissue expansion model in mice, we found that stretch preferentially activates Lgr6+ skin stem cells for skin growth through YAP. By using microarray and single-cell RNA sequencing, we uncovered additional changes in mechanosensitive and metabolic pathways underlying growth control in the skin. This collaborative study between the laboratories of Luis Garza and Sashank Reddy sought to understand the cellular and molecular mechanisms underlying stretch-induced skin regeneration. Our discoveries provide insight into designing future therapies to enhance skin growth for conditions of excess or inadequate skin. Furthermore, the findings here establish a platform for understanding the size dynamics of organs in adult mammals.